Chemically Modified Dendrimers

ABSTRACT

Dendrimers comprising N-acyl urea terminal moieties are described herein. The dendrimers can be used, for example, in the treatment of arthritis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/966,935, filed on Dec. 11, 2015, which is a continuationapplication of U.S. application Ser. No. 14/151,165, filed on Jan. 9,2014, now U.S. Pat. No. 9,283,247, which is a continuation applicationof U.S. application Ser. No. 12/142,266, filed on Jun. 19, 2008, nowU.S. Pat. No. 8,658,148, which claims the benefit of U.S. ProvisionalApplication No. 60/945,815, filed on Jun. 22, 2007, all of which areincorporated herein by reference in their entireties.

TECHNICAL FIELD

This invention relates to dendrimers, compositions comprising thedendrimers, and methods of use thereof, for example, in the treatment ofinflammatory disorders such as arthritis.

BACKGROUND

There are many forms of arthritis, including rheumatoid arthritis,psoriatic arthritis, and osteoarthritis. The most common form ofarthritis, osteoarthritis, is also known as degenerative joint diseaseand can occur following trauma to the joint, following an infection ofthe joint or simply as a result of aging. There is emerging evidencethat abnormal anatomy may contribute to early development ofosteoarthritis.

Osteoarthritis is a condition where low-grade inflammation can result inpain in the joints, for example, pain caused by wearing of the cartilagethat covers and acts as a cushion inside joints. As the bone surfacesbecome less well protected by cartilage, a subject can experience painupon simply bearing weight, for example walking or standing. Due todecreased movement caused by pain, regional muscles may atrophy, andligaments may become more lax.

Arthritis is generally treated with NSAIDs, local injections ofglucocorticoid or viscosupplements based on hyaluronan, salt ofhyaluronic acid or derivative thereof, such as a solution of hyaluronan,and in some cases, with surgery, for example, joint replacement surgery.There is currently no cure for arthritis.

SUMMARY

Applicants have discovered that certain compounds can be used to treatMMP (e.g., MMP-1, -3, or -13) mediated disorders including inflammatorydisorders, such as arthritis. The compounds are dendrimers having aplurality of terminal acyl urea moieties. The term “dendrimer,” whenused herein, refers to a polymer moiety comprised of branched repeatingunits that radiate out from a central atom, multifunctional moiety, orcluster of atoms. Each branched repeating unit terminates with aterminal moiety. An example of a dendrimer is shown below:

wherein I represents the central atom, multifunctional moiety, orcluster of atoms and Z represents the terminal moieties of the branchedrepeating units.

In one aspect, the invention features a dendrimer, the dendrimercomprising a plurality of branched repeating units radiating out from acentral atom, multifunctional moiety, or cluster of atoms, wherein eachrepeating unit comprises a terminal moiety, and wherein at least about5% of the terminal moieties include a urea of formula (I)

wherein

each R¹ and R² is independently C₁-C₆ alkyl, C₁-C₆ alkenyl, cyclyl,aryl, heterocyclyl, heteroaryl, cyclylalkyl, arylalkyl, orheterocyclylalkyl, heteroarylalkyl; any of which are optionallysubstituted by C₁-C₆ alkyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino,hydroxy, C₁-C₆ alkoxy, and wherein either of R¹ or R² independentlyoptionally have a net charge;

indicates the point of attachment of the terminal moiety of formula (I)to the dendrimer; and

wherein less than about 12% of the terminal moieties are covalentlybound to a saccharide moiety.

In some embodiments, the dendrimer comprises a pharmaceuticallyacceptable salt.

In some embodiments, at least about 7% of the terminal moieties includea urea of formula (I) (e.g., at least about 8%, at least about 9%, atleast about 10%, at least about 15%, at least about 20%, or at leastabout 25% of the terminal moieties include a urea of formula (I)).

In some embodiments, less than about 10% of the terminal moieties arecovalently bound to a saccharide moiety (e.g., less than about 8%, lessthat about 6%, less that about 4%, or about 0% of the terminal moietiesare covalently bound to a saccharide moiety).

In some embodiments, either of R¹ or R² has a net positive charge.

In some embodiments, R¹ and R² are both cyclohexyl.

In some embodiments, R¹ and R² are both isopropyl.

In some embodiments, one of R¹ and R² is ethyl and the other of R¹ andR² is dimethylaminopropyl.

In some embodiments, one of R¹ and R² is cyclohexyl and the other of R¹and R² is morpholinoethyl.

In some embodiments, a terminal moiety includes a urea of formula (I)wherein each R¹ and R² is independently C₁-C₆ alkyl and one of R¹ and R²is substituted by C₁-C₆ dialkylamino, and wherein one of R¹ or R² has apositive net charge.

In some embodiments, a terminal moiety includes a urea of formula (I′)

In some embodiments, a terminal moiety includes a urea of formula (I″)

In some embodiments, at least about 5% of the terminal moieties includea urea of formula (I′) or formula (I″) or a combination thereof.

In some embodiments, the dendrimer is selected from the group consistingof a polyamidoamine dendrimer, a polypropylene dendrimer, apolyethyleneimine dendrimer, a carbohydrate based dendrimer, a peptidebased dendrimer, a glycopeptide dendrimer, a metal containing dendrimer,a poly aryl amine dendrimer, a polyamide dendrimer, a poly (alkyl amine)dendrimer, a polyamido alcohol dendrimer, a cyano dendrimer, a polyetherdendrimer, a polythioether dendrimer, a polysiloxane dendrimer, adendritic aryl ester, a perchlorinated dendrimer, a catalytic centercontaining dendrimer, a silicon containing dendrimer, a phosphoruscontaining dendrimer, or a hydrocarbon dendrimer.

In some embodiments, the dendrimer is a polyamidoamine dendrimer.

In some embodiments, at least about 80% of the terminal moieties areterminated with a carboxylate group or with a functionality that can bechemically modified to produce a carboxylate moiety by suitable chemicalreaction.

In some embodiments, the dendrimer is a polyamidoamine dendrimer whereinat least about 50% of the terminal moieties are terminated with acarboxylate group.

In some embodiments, the dendrimer is a polyamidoamine dendrimer of oneof the following generations 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5,8.5, or 9.5. In some embodiments, the dendrimer is a polyamidoaminedendrimer of generation 0.5, 1.5, 2.5, or 3.5 with a carboxylateterminal moiety.

In some embodiments, the dendrimer of formula (I) described herein, ismade by reacting one or more terminal moieties of a starting dendrimerwith a carbodiimide of formula (II) R¹—N═C═N—R², wherein R¹ and R² aredefined above, to provide the dendrimer of formula (I).

In one aspect, the invention comprises a composition comprising theabove-described dendrimer and a pharmaceutically acceptable excipient.

In some embodiments, each dendrimer within the composition comprises atleast about 5% of the terminal moieties including a urea of formula (I).

In some embodiments, the dendrimers, in aggregate within thecomposition, comprise at least about 5% of the terminal moietiesincluding a urea of formula (I).

In some embodiments, the composition further comprises an additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis an anti-inflammatory agent or an analgesic. In some embodiments, theadditional therapeutic agent is a salt of hyaluronic acid or aderivative thereof. In some embodiments, the additional therapeuticagent is a hylan. In some embodiments, the hyaluronic acid is across-linked salt of hyaluronic acid.

In one aspect, the invention features a method of treating or preventingarthritis or reducing a symptom associated with arthritis, the methodcomprising administering to a subject an effective amount of theabove-described dendrimer.

In some embodiments, the method comprises preventing or alleviating oneor more symptoms associated with arthritis or extending the amount oftime prior to the onset of one or more symptoms associated witharthritis.

In some embodiments, the method comprises treating a subject exhibitingone or more symptoms associated with arthritis. In some embodiments, thearthritis is osteoarthritis. In some embodiments, the arthritis isrheumatoid arthritis.

In some embodiments, the method comprises administering an additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis an anti-inflammatory agent or an analgesic. In some embodiments, theadditional therapeutic agent is a salt of hyaluronic acid or aderivative thereof. In some embodiments, the additional therapeuticagent is hylan. In some embodiments, the hyaluronic acid is across-linked hyaluronic acid. In some embodiments, the additionaltherapeutic agent is a mixture of a soluble and crosslinked salt of HAsuch as a mixture of hylan A and hylan B/e.g. Synvisc®.

In some embodiments, the additional therapeutic agent is co-administeredwith the dendrimer. In some embodiments, the additional therapeuticagent is administered before or after administration of the dendrimer.

In some embodiments, the dendrimer is a component in a pharmaceuticalcomposition. In some embodiments, the pharmaceutical compositioncomprises a pharmaceutically acceptable excipient. In some embodiments,the concentration of the dendrimer within the composition is from about1 to about 1000 μg/mL.

In some embodiments, the dendrimer is administered via injection. Insome embodiments, the dendrimer is administered parenterally. In someembodiments, the dendrimer is administered via intraarticular injection.

In some embodiments, the subject is a mammal, for example, a human.

In one aspect, the invention features a method of making theabove-described dendrimer; the method comprising reacting a dendrimercomprising a plurality of terminal carboxylic acid moieties with acarbodiimide, wherein the carbodiimide is substituted with R¹ and R² asdefined anywhere herein, thereby making the dendrimer of formula (I).

In some embodiments, the reaction is performed in aqueous solution. Insome embodiments, the aqueous solution has a pH of from about 4.0 toabout 7.0, for example, a pH of from about 4.5 to about 5.0.

In some embodiments, the reaction is performed in a mixture of water andorganic solvent. In some embodiments, the organic solvent comprises fromabout 5% to less than about 100% by volume. In some embodiments, theorganic solvent comprises about 50% by volume. In some embodiments, theorganic solvent is selected from the group consisting of acetonitrile,tetrahydrofuran, or N-methylpyrrolidone.

In some embodiments, the reaction occurs at a temperature of from about4° C. to about 35° C., for example, at a temperature of from about 20°C. to about 35° C.

In some embodiments, the reaction is maintained for from about 12 toabout 24 hours.

In some embodiments, the method comprises reacting from about 0.5 toabout 10 equivalents of carbodiimide per terminal carboxyl moiety, forexample, from about 1 to about 4 equivalents of carbodiimide perterminal carboxyl moiety.

In some embodiments, the carbodiimide is selected from the groupconsisting of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide,N,N′-Dicyclohexylcarbodiimide, N,N′-Diisopropylcarbodiimide,1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide, andN-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimidemethyl-p-toluenesulfonate, preferablyN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide.

In some embodiments, the method comprises reacting from about 2equivalents of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide perterminal carboxyl moiety.

In some embodiments, the method further comprises purifying the reactionproduct, for example, by dialysis. In some embodiments, the dialysis isperformed at a temperature of from between about 2° C. and about 8° C.In some embodiments, the method further comprises lyophilizing thedialysed product. In some embodiments, the reaction product is purifiedby size exclusion chromatography.

In another aspect, the invention features a dendrimer, the dendrimercomprising a plurality of branched repeating units radiating out from acentral atom, multifunctional moiety, or cluster of atoms, wherein eachrepeating unit comprises a terminal moiety, and wherein at least one ofthe terminal moieties include a urea of formula (I)

in which

each R¹ and R² is independently C₁-C₆ alkyl, C₁-C₆ alkenyl, cyclyl,aryl, heterocyclyl, heteroaryl, cyclylalkyl, arylalkyl, orheterocyclylalkyl, heteroarylalkyl; any of which are optionallysubstituted by C₁-C₆ alkyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino,hydroxy, C₁-C₆ alkoxy, and wherein either of R¹ or R² independentlyoptionally have a net charge;

indicates the point of attachment of the terminal moiety of formula (I)to the dendrimer; and

wherein less than about 12% of the terminal moieties are covalentlybound to a saccharide moiety.

In some embodiments, 0% of the terminal moieties are covalently bound toa saccharide moiety.

In a further aspect, the invention features a dendrimer, the dendrimercomprising a plurality of branched repeating units comprising aplurality of terminal moieties radiating out from a central atom,multifunctional moiety, or cluster of atoms, wherein the dendrimer ismade by reacting at least one terminal moiety with a carbodiimide toprovide a terminal modified dendrimer.

In certain embodiments, the carbodiimide is of formula (II)

R¹—N═C═N—R²  Formula (II)

wherein

each R¹ and R² is independently C₁-C₆ alkyl, C₁-C₆ alkenyl, cyclyl,aryl, heterocyclyl heteroaryl, cyclylalkyl, arylalkyl, orheterocyclylalkyl, heteroarylalkyl; any of which are optionallysubstituted by C₁-C₆ alkyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino,hydroxy, C₁-C₆ alkoxy, and wherein either of R¹ or R² independentlyoptionally have a net charge.

In some embodiments, the reaction is substantially free of saccharidemoieties.

In some embodiments, at least 12% of the terminal moieties react withthe carbodiimide.

As used herein, the term “treat” or “treatment” is defined as theapplication or administration of a dendrimer, alone or in combinationwith, a second compound to a subject, e.g., a patient, or application oradministration of the compound to an isolated tissue or cell, e.g., cellline, from a subject, e.g., a patient, who has a disorder (e.g., adisorder as described herein), a symptom of a disorder, or apredisposition toward a disorder, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisorder, one or more symptoms of the disorder or the predispositiontoward the disorder (e.g., to prevent at least one symptom of thedisorder or to delay onset of at least one symptom of the disorder).

As used herein, an amount of a compound (e.g., a dendrimer) effective totreat a disorder, or a “therapeutically effective amount” refers to anamount of the compound (e.g., a dendrimer) which is effective, uponsingle or multiple dose administration to a subject, in treating a cell,or in curing, alleviating, relieving or improving a subject with adisorder beyond that expected in the absence of such treatment.

As used herein, an amount of an compound (e.g., a dendrimer) effectiveto prevent a disorder, or a “a prophylactically effective amount” of thecompound (e.g., a dendrimer) refers to an amount effective, upon single-or multiple-dose administration to the subject, in preventing ordelaying the occurrence of the onset or recurrence of a disorder or asymptom of the disorder.

As used herein, the term “subject” is intended to include human andnon-human animals. Exemplary human subjects include a human patienthaving a disorder, e.g., a disorder described herein or a normalsubject. The term “non-human animals” of the invention includes allvertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles)and mammals, such as non-human primates, domesticated and/oragriculturally useful animals, e.g., sheep, dog, cat, cow, pig, horse,etc.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a bar graph depicting the effect of dd-NAU and dd-Gln onLevels of Active MMP-1 in chondrocytes stimulated with IL-1β.

FIG. 2 is a bar graph depicting the real-time quantitative PCR analysisof MMP-1 RNA from cells treated with dd-NAU and dd-Gln.

FIG. 3 is a bar graph depicting the real-time quantitative PCR analysisof GAPDH RNA from cells treated with dd-NAU and dd-Gln.

FIG. 4 is a bar graph depicting the real-time quantitative PCR analysisof MMP-1 normalized to GAPDH.

FIG. 5 is a bar graph depicting the effect of dd-NAU and dd-Gln onlevels of active MMP-3 in chondrocytes stimulated with IL-1β.

FIG. 6 is a bar graph depicting the real-time quantitative PCR analysisof MMP-3 RNA from cells treated with dd-NAU and dd-Gln.

FIG. 7 is a bar graph depicting the real-time quantitative PCR analysisof MMP-3 normalized to GAPDH.

FIG. 8 is a bar graph depicting the effects of dd-NAU and dd-Gln onlevels of active MMP-13 in chondrocytes stimulated with IL-1β.

FIG. 9 is a bar graph depicting the real-time quantitative PCR analysisof MMP-3 RNA from cells treated with dd-NAU and dd-Gln.

FIG. 10 is a bar graph depicting the real-time quantitative PCR analysisof MMP-13 normalized to GAPDH.

FIG. 11 depicts histological analysis of bovine cartilage explants.

FIG. 12 is a bar graph depicting the chondroprotective role of dd-NAU(Normalized to Wet Weight).

FIG. 13 is a bar graph depicting the chondroprotective role of dd-NAU(Normalized to DNA).

FIG. 14 is a bar graph depicting the effect of NAU- and glucosaminedendrimers on MMP-1 production by human chondrocytes stimulated withIL-1β.

FIG. 15 is a bar graph depicting the effect of NAU- and glucosaminedendrimers on MMP-3 production by human chondrocytes stimulated withIL-1β.

FIG. 16 is a bar graph depicting the effect of NAU- and glucosaminedendrimers on MMP-13 production by human chondrocytes stimulated withIL-1β.

FIG. 17 is a bar graph depicting the effect of NAU- and glucosaminedendrimers on cell viability in chondrocytes stimulated with IL-1β.

FIG. 18 are histograms depicting the effect of NAU- and glucosaminedendrimers on IL-1α induced proteoglycan loss in bovine cartilageexplant.

FIG. 19 is a bar graph depicting the effect of NAU- and glucosaminedendrimers on IL-1α induced proteoglycan loss in bovine cartilageexplant.

FIG. 20 is a bar graph depicting the effect of NAU- and glucosaminedendrimers on IL-1α induced proteoglycan loss in bovine cartilageexplant.

FIG. 21 are bar graphs depicting the effect of NAU- and glucosaminedendrimers on TNF-α and IL-1β production by human macrophage cell linestimulated with LPS.

FIG. 22 is a bar graph depicting the effect of NAU- and glucosaminedendrimers on IL-6 production by human macrophage cell line stimulatedwith LPS.

FIG. 23 is a bar graph depicting the effect of NAU- and glucosaminedendrimers on cell viability in macrophage cell line stimulated withLPS.

FIG. 24 is a bar graph depicting the effect of Lower generationdendrimers on MMP-1 production by human chondrocytes stimulated withIL-1β.

FIG. 25 are bar graphs depicting the effect of Lower Generation (1.5)dendrimers on MMP-1 production by human chondrocytes stimulated withIL-1β.

FIG. 26 are bar graphs depicting the effect of Lower Generation (1.5)dendrimers on IL-1b production by human THP-1 macrophage cell linestimulated with LPS.

DETAILED DESCRIPTION

Dendrimer Compounds

Dendrimers are well-defined macromolecules that have a specific size,shape, and chemical functionality. A dendrimer is a branchedmonodisperse macromolecular compound. Structurally dendrimers are highlybranched macromolecules that can be subdivided into three architecturalcomponents: a central core branched cell, interior branch cell andbranch cell possessing surface groups. They are generally synthesizedthrough a stepwise repetitive reaction sequence. The dendrimersdescribed herein include a plurality of terminal moieties comprising anN-acyl urea moiety as provided below:

Each R¹ and R² is independently C₁-C₆ alkyl, C₁-C₆ alkenyl, cyclyl,aryl, heterocyclyl, heteroaryl, cyclylalkyl, arylalkyl, orheterocyclylalkyl, heteroarylalkyl; any of which are optionallysubstituted by C₁-C₆ alkyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino,hydroxy, C₁-C₆ alkoxy, and wherein either of R¹ or R² independentlyoptionally have a net charge;

indicates the point of attachment of the terminal moiety of formula (I)to the dendrimer. In preferred embodiments, at least 5% of the terminalmoieties have the structure provided above (e.g., at least about 7%, 8%,9%, 10%, 15%, 20%, or 25%). In some embodiments, one or more terminalmoieties of the dendrimer not having the formula above are carboxymoieties (for example, all of the terminal moieties not having theformula above are carboxy moieties). The dendrimers described hereinhave less than 12% of the terminal moieties covalently bound to asaccharide moiety, for example Gln (e.g., less than about 10%, 8%, 6%,4%, or 1%). In some preferred embodiments, none of the terminal moietiesare covalently bound to a saccharide moiety.

In some preferred embodiments at least one of R¹ and R² includes anitrogen moiety (e.g., an alkyl substituted with an amino moiety or anitrogen containing heterocyclic moiety such as piperidine ormorpholine). For example, one of R¹ and R² has a net positive charge.

In some preferred embodiments one of R¹ and R² is an unsubstituted alkylmoiety (e.g., C₁-C₆ alkyl, preferably C₁-C₃ alkyl, e.g., methyl orethyl) and the other of R¹ or R² is an alkyl substituted with an aminomoiety (e.g., a dialkyl amino moiety such as dimethylamino). Forexample, one of R¹ and R² is ethyl and the other of R¹ or R² isdimethylaminopropyl or one of R¹ and R² is cyclohexyl and the other ofR¹ and R² is morpholinoethyl. In some instances where one or R¹ and R²includes a nitrogen moiety the terminal moiety forms a salt (e.g., withthe cationic amine nitrogen, for example, to produce a quaternaryamonium).

Some preferred terminal moieties are provided below:

In some embodiments neither of R¹ or R² includes a heteroatomsubstituent. For example, in some embodiments, R¹ and R² are bothisopropyl or R¹ and R² are both cyclohexyl.

Examples of dendrimers include the following: a polyamidoaminedendrimer, a polypropylene dendrimer, a polyethyleneimine dendrimer, acarbohydrate based dendrimer, a peptide based dendrimer, a glycopeptidedendrimer, a metal containing dendrimer, a poly aryl amine dendrimer, apolyamide dendrimer, a poly (alkyl amine) dendrimer, a polyamido alcoholdendrimer, a cyano dendrimer, a polyether dendrimer, a polythioetherdendrimer, a polysiloxane dendrimer, a dendritic aryl ester, aperchlorinated dendrimer, a catalytic center containing dendrimer, asilicon containing dendrimer, a phosphorus containing dendrimer, or ahydrocarbon dendrimer. The preferred dendrimers include a polyvalentcore covalently bonded to at least two dendritic branches. Particularlypreferred dendrimers are dendrimers where the core and interiorsbranches are derived from cells. Exemplary preferred embodiments arepolyamidoamine (PAMAM) dendrimers (e.g., generation 1.5, 2.5, or 3.5),PAMAM (EDA) dendrimers, polylysine dendrimers, polypropylene dendrimerand the branch cell containing surface group carboxylic terminalmoieties.

In some preferred embodiments, the dendrimer is a PAMAM generation 3.5dendrimer wherein from about 8 to about 15% of the terminal moietieshave one of the following structures:

Methods of Making Dendrimers

The preparation of dendrimers is discussed in U.S. Pat. Nos. 4,507,466,4,558,120, 4,568,737 and 4,587,329 (PAMAM dendrimers), as well as inU.S. Pat. Nos. 4,289,872 and 4,410,688 (lysine based dendrimer).International Patent Publications Nos. WO 88/01178, WO 88/01179 and WO88/01180 disclose conjugates or associates of dendrimer with anothermaterial such as a carried pharmaceutical material.

The dendrimers described herein are prepared by reacting at least aportion of the terminal moieties (e.g., terminal carboxy moieties) witha reactant such as a carbodiimide, which produces some N-acyl urea. Insome embodiments, not all of the terminal moieties react with thereactant (e.g., carbodiimide (i.e., “CDI”)) to provide an N-acyl urea,but instead remain unreacted carboxy moieties. Thus, the dendrimersdescribed herein generally include terminal moieties with at least twostructural variations (e.g., the starting structure of the terminalmoiety and the structure of the reacted product (i.e., N-acyl urea)). Ininstances where the dendrimer is reacted with an unsymmetrical reactant(e.g., an unsymmetrical carbodiimide), then the dendrimer can includethree different structural variations on the terminal moieties. In someembodiments, the starting terminal moiety is reacted with a CDI toprovide yet a modified terminal moiety other than the starting materialor the final N-acyl urea.

Exemplary carbodiimide reactants includeN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide,N,N′-Dicyclohexylcarbodiimide, N,N′-Di isopropylcarbodiimide,1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide methiodide, andN-Cyclohexyl-N′-(2-morpholinoethyl)carbodiimidemethyl-p-toluenesulfonate. In some preferred embodiments,N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide is reacted with thedendrimer.

The degree of reaction of the reactant (e.g., carbodiimide) with thestarting terminal moieties can be controlled or varied by modificationof the reaction conditions. For example, an increase in molarequivalents of carbodiimide to terminal carboxyl moiety generallyincreases the relative amount of terminal moieties on the startingdendrimer (e.g., carboxy moieties) that are converted (e.g., to N-acylurea moieties). Exemplary ratios are from about 0.5 to about 10 molarequivalents of carbodiimide to terminal moieties (e.g., from about 1 toabout 4, e.g., about 2 or about 4). In general, at least one terminalmoiety is reacted.

The reaction can be further controlled with conditions such astemperature, pH, solvent and time. The dendrimers are generally reactedwith starting reagent at a temperature of from about 4° C. to about 35°C. (e.g., from about 20° C. to about 35° C.). In instances where thedendrimers are formed using a carbodiimide, the pH of the reaction isgenerally from about 4.5 to about 7.0, e.g., from about 4.5 to about5.0. The reactions are generally performed in an aqueous solution. Insome embodiments, the reactions are performed in a mixture of water andan organic solvent, for example, acetonitrile, tetrahydrofuran, orN-methylpyrrolidone. In general, the starting materials are subjected toreaction conditions for about 12-24 hours.

After the starting dendrimer has been treated with reagent, the reactionproduct is generally purified, for example, using precipitation,chromatography (e.g., ion-exchange), dialysis or filtration. Thepurified product can be further processed, for example lyophilized.

Dendrimer products can be characterized by various analytical techniquesto determine the degree of terminal modification. These assays includeHPLC, RPLC, UPLC, CZE, and CGE separation techniques in combination withdetection methods using MALDI-TOF, LIF, UV, CAD or ESI-MS. Additionally,1H NMR analysis is capable of characterizing the dendrimer products.

Methods of Using Dendrimers

In some embodiments, a dendrimer described herein is administered to asubject to reduce one or more symptoms or manifestations of arthritis.For example, a dendrimer described herein can modify (e.g., reduce) andpain and/or other symptoms associated with arthritis. Clinical endpointsthe can be used to evaluate the modification of one or more of pain orsymptoms include X-ray (e.g., evaluation of Joint space narrowing), MRI(for evaluation of synovitis (inflammation cell infiltration),evaluation of cartilage volume and proteoglycan content),Arthroscopy/Ultrasonography (evaluation of visualize cartilage lesion),Biomarkers (evaluation of serum, urine and synovial fluid analyzed forcollagen type I and II breakdown products, MMP-1, 3, 13, IL-1, IL-6,TNFa, COMP-1) and evaluation of Pain scores for symptoms.

The dendrimers described herein have been found to exhibit activityagainst MMP activity (e.g., MMP-1, -3, and -13), which has beenimplicated in arthritis (e.g., osteoarthritis or rheumatoid arthritis).The dendrimers described herein can be used to treat, prevent, or delaythe onset of an inflammatory disorder such as arthritis (e.g.,osteoarthritis or rheumatoid arthritis).

The dendrimers described herein can, for example, be administered byinjection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, subcutaneously, or intraarticularly(e.g., in the joint space); or orally, buccally, nasally,transmucosally, topically, in an ophthalmic preparation, or byinhalation, with a dosage ranging from about 0.001 to about 100 mg/kg ofbody weight, e.g., between 0.001-1 mg/kg, 1-100 mg/kg, or 0.01-5 mg/kg,every 4 to 120 hours, e.g., about every 6, 8, 12, 24, 48, or 72 hours,or according to the requirements of the particular dendrimer. Themethods herein contemplate administration of an effective amount ofdendrimer or dendrimer composition to achieve the desired or statedeffect (e.g., reduction of pain and/or inflammation in a subject).Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to about 6 times per day. Alternatively, thedendrimers can be administered as a continuous infusion. Suchadministration can be used as a chronic or acute therapy. The amount ofactive ingredient that may be combined with the carrier materials toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. A typical preparation willcontain from about 5% to about 95% active compound (w/w). Alternatively,such preparations contain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificdendrimer employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of adendrimer, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of disease symptoms.

In some embodiments a dendrimer described herein is administered withanother therapeutic agent, for example an anti-inflammatory agent or ananalgesic. As used herein, “co-administration” or “combination therapy”means that two or more agents are administered to a subject at the sametime or within an interval such that there is an overlap of an effect ofeach agent on the patient. In some embodiments, agents are administeredwithin 15, 10, 5, or 1 minute of one another. Preferably, theadministrations of the agents are spaced sufficiently close togetherthat such a combinatorial effect is achieved. The agents can beadministered simultaneously, for example, in a combined unit dose(providing simultaneous delivery of both agents). Alternatively, theagents can be administered at a specified time interval, for example, aninterval or minutes, hours, days, or weeks. In general, however, theagents are concurrently bioavailable, e.g., detectable in the subject.

Exemplary anti-inflammatory agents include glucocorticoids and NSAIDS.Exemplary NSAIDS include salicylates, arylakanoic acids, 2-arylpropionicacids (profens), N-arylanthranilic acids (fenamic acids), pyrazolidinederivatives, oxicams, COX2 inhibitors, and sulphonanalides. Exemplaryanti-inflammatory compounds include the following: Anti-inflammatorycompounds include but are not limited to Alclofenac; AlclometasoneDipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide;Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac;Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen;Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide;Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate;Clobetasone Butyrate; Clopirac; Cloticasone Propionate; CormethasoneAcetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone;Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium;Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate;Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab;Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole;Fenbufen; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin;Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride Lornoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone;Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;Paranyline Hydrochloride; Pentosan Polysulfate Sodium; PhenbutazoneSodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin;Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate;Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide;Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium;Triclonide; Triflumidate; Zidometacin; Zomepirac Sodium. In someembodiments, a dendrimer described herein is combined with an analgesic.Analgesics are a class of drugs that includes most painkillers, such asaspirin, paracetamol (acetaminophen), and ibuprofen, e.g., andadditional agents listed herein. Other examples include the nonsteroidalanti-inflammatory drugs (NSAIDs) such as salicylates, COX2 inhibitors,narcotic drugs such as morphine, and synthetic drugs with narcoticproperties such as tramadol. Acetaminophen (Tylenol), Codeine (Tylenol#2,3,4), Darvocet (Propoxyphene/Acetaminophen), Darvon (Propoxyphene),Duragesic (Fentanyl Patch), Hydromorphone (Palladone, Dilaudid,)Morphine (MSContin, Oramorph), Oxycodone (OxyContin, Roxicodone),Percocet (Oxycodone/Acetaminophen), Percodan (Oxycodone/Aspirin), TalwinNX (Pentazocine/Naloxone), Ultracet (Tramadol/Acetaminophen), Ultram(Tramadol), and Vicodin (Hydrocodone/Acetaminophen).

In some embodiments, a dendrimer described herein is combined with asalt of hyaluronic acid or a derivative thereof, for example, hylan, across-linked hyaluronic such as Synvisc or Hylastan, Seprafilm, orcombinations thereof. (See, for example, U.S. Pat. Nos. 4,582,865;4,713,448; 5,153,724; 5,099,013; 6,521,223, 5,017,229, 5,827,937,7,226,972, and 6,921,819; and PCT publication WO 2005/066215 each ofwhich is incorporated herein by reference.)

Dendrimer Containing Compositions

Pharmaceutical compositions of this invention comprise a dendrimerdescribed herein or a pharmaceutically acceptable salt thereof; anadditional compound including for example, an anti-inflammatory (e.g., asteroid) or an analgesic; and any pharmaceutically acceptable carrier,adjuvant or vehicle.

Compositions of this invention comprise a dendrimer described herein ora pharmaceutically acceptable salt thereof (e.g., where one or moreterminal moieties of the dendrimer is a pharmaceutically acceptablesalt); and a pharmaceutically acceptable carrier, adjuvant or vehicle.The compositions described herein include the dendrimers describedherein, as well as additional therapeutic compounds if present, inamounts effective for achieving a modulation of disease or diseasesymptoms, including MMP mediated disorders or symptoms thereof. Thecompositions are made by methods including the steps of combining one ormore dendrimers described herein with one or more carriers and,optionally, one or more additional therapeutic compounds describedherein.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha dendrimer described of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase which can be combined with emulsifying and/orsuspending agents. If desired, certain sweetening and/or flavoringand/or coloring agents may be added.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. In someembodiments, a dendrimer described herein is combined with a salt ofhyaluronic acid or a derivative thereof, for example, hylan, across-linked hyaluronic acid such as Synvisc or Hylastan, Seprafilm, orcombinations thereof. (See, for example, U.S. Pat. Nos. 4,582,865;4,713,448; 5,153,724; 5,099,013; 6,521,223, 5,017,229, 5,827,937,7,226,972, and 6,921,819; and PCT publication WO 2005/066215 each ofwhich is incorporated herein by reference.) The sterile injectablepreparation may also be a sterile injectable solution or suspension in anon-toxic parenterally acceptable diluent or solvent, for example, as asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed are mannitol, water, Ringer's solution and isotonicsodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms such as emulsions and or suspensions. Other commonly usedsurfactants such as Tweens or Spans and/or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asD-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, may also beadvantageously used to enhance delivery of compounds of the formulaedescribed herein.

In some cases, the pH of the formulation may be adjusted withpharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated compound or its delivery form.

The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intraarticular,intraarterial, intrasynovial, intrasternal, intrathecal, intralesional,and intracranial injection or infusion techniques.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When the compositions of this invention comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalcompound should be present at dosage levels of between about 1 to 100%,and more preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. Additionally, combinations of aplurality of compounds described herein are also envisioned. Theadditional compounds may be administered separately, as part of amultiple dose regimen, from the compounds of this invention.Alternatively, those compounds may be part of a single dosage form,mixed together with the compounds of this invention in a singlecomposition.

EXAMPLES

Examples 1 to 10 provide methods of making a dendrimer, for example, adendrimer described herein. Exemplary dendrimers include modified PAMAMdendrimers such as generations 3.5, 2.5, and 1.5.

Example 1: Treatment of PAMAM Dendrimer Generation 3.5 with EDC withDialysis Purification

A solution of 10% w/w PAMAM Dendrimer Generation 3.5 in methanol (6.0 g,2.97 mmol COOH) was transferred into a 50 mL round bottom flask.Methanol was removed by rotary evaporation. A solution of1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.139 g,5.94 mmol) in water (30 mL) was prepared. The EDC solution was added tothe flask containing the dendrimer. The initial pH of the solution wasmeasured at 8.83. The pH was adjusted with 1N HCl to 5.04. The reactionwas clear and colorless. The reaction was allowed to go overnight withstirring at room temperature. At the completion of the reaction, thesolution was loaded into dialysis tubing (SpectraPor 1000 MWCO) anddialyzed against PBS (two times) and against sterile water for injection(two times) over a period of several days to remove any EDU. Thecontents of the dialysis tubing were recovered by freeze drying. Theproduct was a white, fine solid. Yield=0.4215 g.

Example 2: Treatment of PAMAM Dendrimer Generation 3.5 with EDC withoutDialysis Purification

A solution of 10% w/w PAMAM Dendrimer Generation 3.5 in methanol (5.0mL, 2.48 mmol COOH) was transferred into a 50 mL conical tube. Methanolwas allowed to evaporate overnight at room temperature. Water (8 mL) wasadded to the dried dendrimer. The conical tube was capped and placed ona rotator for 30 minutes. The resulting solution was homogeneous. The pHof the solution was between 9.5-10.0. The pH was adjusted by adding 2NHCl to pH of 4.5-5.0. A stock solution of1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (9.573 g,49.9 mmol) in water (220 mL) was prepared. To the dendrimer solution wasadded 22.0 mL of the EDC stock solution corresponding to 4.96 mmol ofEDC. The pH of the final reaction solution required additionaladjustment to pH 4.5-5.0 with 2N HCl. The reaction was clear andcolorless. The conical tube was capped and placed on a rotator overnightat room temperature. At the completion of the reaction, the solution wasfiltered using an Acrodisc Mustang E membrane at a rate of 1.0 mL perminute. The solution was aliquoted and stored at 2-8° C.

Example 3: Treatment of PAMAM Dendrimer Generation 3.5 with EDC in thePresence of Half Equivalent of Glucosamine

A solution of 10% w/w PAMAM Dendrimer Generation 3.5 in methanol (6.0 g,2.97 mmol COOH) was transferred into a 50 mL round bottom flask.Methanol was removed by rotary evaporation. A solution of Glucosaminehydrochloride (0.320 g, 1.48 mmol) in water (8 mL) was prepared andadded to the flask. The pH of the reaction solution was adjusted to 5.0with 1N HCl. A solution of1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.139 g,5.94 mmol) in water (23 mL) was prepared. The EDC solution was added tothe flask. The solution pH was measured and did not require adjustment.The reaction was clear and colorless. The reaction was allowed to go for2 hours with stirring at room temperature. After 2 hours, 18 mL of thereaction solution was transferred into dialysis membrane (SpectraPor1000 MWCO). The remaining reaction solution was allowed to go overnightwith stirring at room temperature. At the completion of the reaction,the remaining solution was loaded into dialysis tubing (SpectraPor 1000MWCO) and both samples were dialyzed against NaCl (two times) andagainst sterile water for injection (two times) over a period of severaldays to remove any EDU. The contents of the dialysis tubing wererecovered by freeze drying. The product was a white, fine solid. YieldB1=0.2619 g; Yield B2=0.0112 g.

Example 4: Treatment of PAMAM Dendrimer Generation 3.5 with EDC in thePresence of Half Equivalent of Glucosamine

A solution of 10% w/w PAMAM Dendrimer Generation 3.5 in methanol (6.0 g,2.97 mmol COOH) was transferred into a 50 mL round bottom flask.Methanol was removed by rotary evaporation. A solution of Glucosaminehydrochloride (0.320 g, 1.48 mmol) in water (8 mL) was prepared andadded to the flask. The pH of the reaction solution was adjusted to 5.0with 1N HCl. A solution of1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.139 g,5.94 mmol) in water (22 mL) was prepared. The EDC solution was added tothe flask. The solution pH was measured and adjusted to 4.5-5.0 with 1NHCl. The reaction was clear and colorless. The reaction was allowed togo for 2 hours with stirring at room temperature. After 2 hours, 15 mLof the reaction solution was transferred into dialysis membrane(SpectraPor 1000 MWCO). The remaining reaction solution was allowed togo overnight with stirring at room temperature. At the completion of thereaction, the remaining 15 mL of solution was loaded into dialysistubing (SpectraPor 1000 MWCO) and both samples were dialyzed against PBS(one time), NaCl (one time), and finally against sterile water forinjection (one time) over a period of several days to remove any EDU.The contents of the dialysis tubing were recovered by freeze drying. Theproduct was a white, fluffy solid. Yield C1=0.3007 g; Yield C2=0.3972 g.

Example 5: Treatment of PAMAM Dendrimer Generation 1.5 with EDC

A solution of 20% w/w PAMAM Dendrimer Generation 1.5 in methanol (˜2.9mL, 2.73 mmol COOH) was transferred into 4×50 mL conical tube. Methanolwas allowed to evaporate overnight at room temperature. Water was addedto the dried dendrimer. To reaction 78-1A was added 30 mL water, to78-1B was added 25 mL, to 78-1C was added 20 mL, and to 78-1D was added10 mL water. The conical tubes were capped and placed on a rotator for30 minutes. The resulting solution was homogeneous. The pH of thesolutions was between 9.5-10.0. The pH was adjusted by adding 2N HCl topH of 4.5-5.0. A stock solution of1-[3-(Dimethylamino)propyl]-3-cthylcarbodiimide hydrochloride (20.9 g,0.109 mol) in water (100 mL) was prepared. To the reaction solution wasadded a volume of the EDC stock solution. To reaction 78-1A was added 0mL of EDC solution, to 78-1B was added 5 mL EDC solution (5.45 mmol), to78-1C was added 10 mL (10.9 mmol), and to 78-1D was added 20 mL (21.8mmol). These volumes of the EDC solution correspond to 0 eq, 2 eq, 4 eq,and 8 eq of EDC. The pH of the final reaction solution requiredadditional adjustment to pH 4.5-5.0 with 2N HCl. The reaction was clearand colorless. The conical tubes were capped and placed on a rotatorovernight at room temperature. At the completion of the reaction, thesolutions were filtered using an Acrodisc Mustang E membrane at a rateof 1.0 mL per minute. The solutions were aliquoted and stored at 2-8° C.

Example 6: Treatment of PAMAM Dendrimer Generation 2.5 with EDC

A solution of 10% w/w PAMAM Dendrimer Generation 1.5 in methanol (˜6.3mL, 2.554 mmol COOH) was transferred into 4×50 mL conical tube. Methanolwas allowed to evaporate overnight at room temperature. Water was addedto the dried dendrimer. To reaction 78-2A was added 30 mL water, to78-2B was added 25 mL, to 78-2C was added 20 mL, and to 78-2D was added10 mL water. The conical tubes were capped and placed on a rotator for30 minutes. The resulting solution was homogeneous. The pH of thesolutions was between 9.5-10.0. The pH was adjusted by adding 2N HCl topH of 4.5-5.0. A stock solution of1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (20.9 g,0.109 mol) in water (100 mL) was prepared. To the reaction solution wasadded a volume of the EDC stock solution. To reaction 78-1A was added 0mL of EDC solution, to 78-1B was added 5 mL EDC solution (5.45 mmol), to78-1C was added 10 mL (10.9 mmol), and to 78-1D was added 20 mL (21.8mmol). These volumes of the EDC solution correspond to roughly 0 eq, 2eq, 4 eq, and 8 eq of EDC. The pH of the final reaction solutionrequired additional adjustment to pH 4.5-5.0 with 2N HCl. The reactionwas clear and colorless. The conical tubes were capped and placed on arotator overnight at room temperature. At the completion of thereaction, the solutions were filtered using an Acrodisc Mustang Emembrane at a rate of 1.0 mL per minute. The solutions were aliquotedand stored at 2-8° C.

Example 7: Treatment of PAMAM Dendrimer Generation 3.5 with EDC Followedby Dialysis Purification

Synthesis of N-Acyl Urea Derivative of PAMAM Dendrimer Generation 3.5. Asolution of 10% w/w PAMAM Dendrimer Generation 3.5 in methanol (9.0 g,4.46 mmol COOH) was transferred into a 125 mL nalgene container.Methanol was allowed to evaporate overnight at room temperature. Sterilewater for irrigation (15 mL) was added to the container. The solutionwas mixed at room temperature for 2 hours. The solution was clear,colorless, and homogeneous. The solution pH was measured and adjusted to4.5-5.0 with 2N HCl. A solution of1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (2.277 g,11.88 mmol) in water (60 mL) was prepared. The EDC solution (45 mL),corresponding to 8.91 mmol of EDC, was added to the flask containing thedendrimer. The pH was measured at 4.5-5.0 and did not requireadjustment. The reaction was allowed to go overnight with stirring atroom temperature. At the completion of the reaction, the solution wasloaded into dialysis tubing (SpectraPor 3500 MWCO) and dialyzed againstNaCl (two times) and against sterile water for injection (two times)over a period of several days to remove any EDU. The contents of thedialysis tubing were recovered by freeze drying. The product was a whitesolid. Yield=0.8184 g.

Example 8: Treatment of PAMAM Dendrimer Generation 3.5 with EDC in thePresence of AlexaFluor 488 Cadaverine

A solution of 10% w/w PAMAM Dendrimer Generation 3.5 in methanol (1.0 g,0.50 mmol COOH) was transferred into a 20 mL scintillation vial.Methanol was allowed to evaporate overnight at room temperature. Sterilewater for irrigation (3.0 mL) was added to the vial. The solution wasmixed at room temperature for 2 hours. The solution was clear,colorless, and homogeneous. The solution pH was measured and adjusted to4.5-5.0 with 2N HCl. Water (1000 uL) was added to 2×1 mg of AF488. TheAF488 solutions were dark green and were transferred into the reactionvial. The pH was measured at 4.68. A solution of1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (2.277 g,11.88 mmol) in water (60 mL) was prepared. The EDC solution (5.0 mL),corresponding to 0.99 mmol of EDC, was added to the flask containing thedendrimer. The pH was measured at 4.5-5.0 and did not requireadjustment. The reaction was allowed to go overnight at room temperaturewhile protected from light. At the completion of the reaction, thesolution was loaded into dialysis tubing (SpectraPor 3500 MWCO) anddialyzed against NaCl (two times) and against sterile water forinjection (two times) over a period of several days to remove any EDU.The contents of the dialysis tubing were recovered by freeze drying. Theproduct was a pink/orange solid. Yield=0.0881 g.

Example 9: Treatment of PAMAM Dendrimer Generation 3.5 with EDC and Halfan Equivalent of Glucosamine without Dialysis Purification

A solution of 10% w/w PAMAM Dendrimer Generation 3.5 in methanol (5.0mL, 2.48 mmol COOH) was transferred into a 50 mL conical tube. Methanolwas allowed to evaporate overnight at room temperature. A solution ofGlucosamine hydrochloride (0.267 g, 1.24 mmol) in water (8 mL) wasprepared and added to the flask. The conical tube was capped and placedon a rotator for 30 minutes. The resulting solution was homogeneous. ThepH of the solution was between 7.5-8. The pH was adjusted by adding 2NHCl to pH of 4.5-5.0. A stock solution of1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (9.573 g,49.9 mmol) in water (220 mL) was prepared. To the dendrimer solution wasadded 22.0 mL of the EDC stock solution corresponding to 4.96 mmol ofEDC. The pH of the final reaction solution required additionaladjustment to pH 4.5-5.0 with 2N HCl. The reaction was clear andcolorless. The conical tube was capped and placed on a rotator overnightat room temperature. At the completion of the reaction, the solution wasfiltered using an Acrodisc Mustang E membrane at a rate of 1.0 mL perminute. The solution was aliquoted and stored at 2-8° C.

Example 10: Treatment of PAMAM Dendrimer Generation 3.5 with EDC

A solution of 10% w/w PAMAM Dendrimer Generation 3.5 in methanol (5.0 g,2.48 mmol COOH) was transferred into 6×125 mL nalgene containers.Methanol was allowed to evaporate overnight at room temperature. Waterwas added to the dried dendrimer. To reactions 150-1 and 150-2 was added30 mL water, to 150-3 was added 28 mL water, to 150-4 was added 25 mL,to 150-5 was added 20 mL, and to 150-6 was added 10 mL water. Thecontainers were closed and mixed for 30 minutes. The resulting solutionwas homogeneous. The pH of the solutions was between 9.5-10.0. The pHwas adjusted by adding 2N HCl to pH 4.5-5.0. A stock solution of1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (9.49 g,4.95 mmol) in water (50 mL) was prepared. To the reaction solution wasadded a volume of the EDC stock solution. To reaction 150-1 was added625 uL of EDC solution (0.62 mmol), to 150-2 was added 1250 uL EDCsolution (1.24 mmol), to 150-3 was added 2500 uL EDC solution (2.48mmol), to 150-4 was added 5 mL (4.96 mmol), to 150-5 was added 10 mL(9.92 mmol), and to 150-6 was added 20 mL (19.84 mmol). These volumes ofthe EDC solution correspond to roughly 0.25, 0.5 eq, 1.0 eq, 2 eq, 4 eq,and 8 eq of EDC. The pH of the final reaction solution was between pH4.5-5.0 and did not require adjustment. The reaction was clear andcolorless. The containers were closed and allowed to sit overnight atroom temperature. At the completion of the reaction, the solutions wereloaded into dialysis tubing (SpectraPor 3500 MWCO) and dialyzed againstNaCl (two times) and against sterile water for injection (two times)over a period of several days to remove any EDU. The contents of thedialysis tubing were recovered by freeze drying. The product was a whitesolid. Yield=N/A.

Example 11: Evaluation of Terminal Modified PAMAM Generation 3.5Dendrimers In Vitro

The effects of dendrimer compounds comprising n-acyl urea terminalmoieties (i.e., dd-NAU) were evaluated for MMP activity in chondrocytes.The materials were prepared using methods described in Examples 2-9. Insome instances, these effects were compared to dendrimer compoundscomprising glucosamine terminal moieties (i.e., dd-Gln) and/or ethyldimethylaminopropylurea (EDU). Chondrocytes were grown to confluency in6-well plates (4 donors). The chondrocytes were pretreated withdendrimers (e.g., dd-NAU or dd-Gln) or EDU for 2 hours and thenstimulated with IL-1β for 24 hours. Supernatants were collected andMMP-1, -3, and 13 ELISA assays were performed. The cells were collectedfor RNA analysis (3 donors).

The results of the assays are provided in FIGS. 1-10. FIGS. 1, 5, and 8depict the effects of dd-NAU and dd-Gln on levels of active MMP-1, -3,and 13 respectively. These results are also compared to cells treatedwith EDU. FIGS. 2 and 6 depict the results of real-time PCR analysis ofMMP-1 RNA and MMP-3 RNA respectively of cells treated with dd-NAU anddd-Gln. Real time PCT was also used to evaluate GAPDH RNA and MMP-3 RNAfrom cells treated with dd-NAU and dd-Gln the results of which are shownin FIG. 3 and FIG. 9 respectively. FIGS. 4, 7, and 10 depict theanalysis of MMP-1, -3, and 13 respectively to normalized to GAPDH. IC₅₀constants for inhibition of MMP activity is provided in Table 1 below:

TABLE 1 Glucos- N-Acetyl amine NAU Glucosamine glucosamine DendrimerDendrimer EDU (μM) (μM) (μM) (μM) (μM) MMP-1 1420 N/A N/A 16 N/A MMP-3600 N/A N/A 17 N/A MMP-13 1400 N/A N/A 10 N/A N/A = did not affect MMPlevels

IC₅₀ constants for inhibition of MMP mRNA levels are provided in Table 2below:

TABLE 2 NAU Dendrimer Glucosamine NAU (μM) Dendrimer Dendrimer Protein(μM) (μM) MMP-1 16 N/A 15 MMP-3 17 N/A 15 MMP-13 10 N/A 10 N/A = did notaffect MMP levels

Example 12: Evaluation of Terminal Modified PAMAM Generation 3.5Dendrimers on Cartilage

The effects of dendrimer compounds comprising N-acyl urea terminalmoieties (i.e., dd-NAU) parepared as described in Example 2 wereevaluated on cartilage explants stimulated with IL-1β. Cartilageexplants (6 mm punch) weighing 60-80 mg were prepared. The explants werestimulated with IL-1β in the presence of dd-NAU dd-Gln prepared asdescribed in Example 9 or CMC for four days. Histological analysis wasperformed on the explants based on the procedures disclosed in Nishidaet al., Osteoarthritis Cartilage 2004 May 12(5):374-82; and Soder etal., Arthritis Rheum. 2005 February 52(2):468-78.

FIG. 11 demonstrates the protective effects of dd-NAU on bovinecartilage relative to those treated with dd-Gln or CMC as provided byhistological analysis.

FIGS. 12 and 13 demonstrate a chondroprotective role of dd-NAU asdemonstrated in the normalized to wet weight of the cartilage (FIG. 12)and normalized to DNA (FIG. 13). These results show a dose response forchondroprotection with dd-NAU.

Example 13: Evaluation of Terminal Modified PAMAM Generation 3.5Dendrimers In Vitro

Chondrocytes were grown to confluency in 6-well plates (2 donors). Thechondrocytes were then pretreated with either a dendrimer as prepared inExample 2, a dd-Gln dendrimer prepared as described in Example 9, or anunmodified dendrimer. Supernatants were collected and MMP-1, -3, and 13ELISA assays were performed. Neutral Red assays and Calcien AM assayswere also performed on the chondrocytes to determine cell viability.

FIGS. 14-16 respectively demonstrate the effect of dd-NAU and dd-Gln onMMP-1, -3, and 13, production by human chondrocytes stimulated withIL-1β. FIG. 17 demonstrates the effect of the assayed compounds on cellviability. As seen by the data, cell viability remains high in cellstreated with the assayed compounds.

Example 14: Evaluation of Terminal Modified PAMAM Generation 3.5Dendrimers in Cartilage

Bovine cartilage explants (6 mm punch) weighing between 60 and 80 mgswere prepared. The explants were pretreated with one of dd-NAU preparedas described in, dd-Gln, EDU, or unmodified dendrimer for two hours. Thedendrimers were prepared as described in Example 13. The cartilage wasthen stimulated with IL-1β (5 ng/ml) for four days. Histological andbiochemical analysis were then performed on the samples.

FIG. 18 demonstrates the protective effect of the assayed compoundsbased on a histological analysis.

FIGS. 19 and 20 demonstrate the effect of dd-NAU, dd-Gln, EDU, orunmodified dendrimer on IL-1α induced proteoglycan loss in bovinecartilage explants.

Example 15: Evaluation of Terminal Modified PAMAM Generation 3.5Dendrimers on LPS Induced Cytokine Production and Cell Viability in aTHP-1 Macrophage Cell Line

A THP-1 cell line (1E6) was plated in 24-wells. The cells were activatedwith PMA for 24 hours. The cells were then treated with one of dd-NAU,dd-Gln (made as described in Example 9), EDU, or unmodified dendrimerfor two hours. The cells were then stimulated with LPS for 24 hours.Supernatants were then analyzed for cytokines.

FIGS. 21 and 22 demonstrate the effect of the assayed compounds on TNF-αand IL-1β production by human macrophages stimulated with LPS. dd-NAUinhibited LPS induced upregulation of pro-inflammatory cytokines (IL-1β,IL-6 and TNF-α) without adversely affecting cell viability as shown FIG.23. FIG. 23 demonstrates the effect of the assayed compounds on cellviability. As seen by the data, cell viability remains high in cellstreated with the assayed compounds.

Example 16: Evaluation of Terminal Modified Dendrimers In Vitro UsingPAMAM Generation 1.5 and 2.5 Modified Dendrimers

Modified dendrimers as prepared in Examples 5 and 6 were assayed usingthe protocols described in Example 13 for the effect of the compounds onMMP-1 production by human chondrocytes stimulated with IL-1β. Thesecompounds were compared to those modified dendrimers as preparedaccording to Example FIG. 24 depicts the comparative effect of themodified PAMAM dendrimer of generation 1.5 with that of generation 2.5.As can be seen in the figures, both generations of dendrimers inhibitedIL-1β induced MMP production. The levels of inhibition was similar tothe level seen with the dendrimers prepared according to Example 2. Thedendrimers also demonstrated an increase in potency by increasing theamount of EDA in the chemical reaction, for example, as provided in FIG.25.

IC₅₀ values for the modified dendrimers are provided in Tables 3, 4, and5 provided in the Appendix.

Example 17: Evaluation of Terminal Modified PAMAM Generation 1.5 and 2.5Dendrimers on LPS Induced Cytokine Production and Cell Viability in aTHP-1 Macrophage Cell Line

THP-1 cells were prepared and assayed as described in Example 15. FIG.26 demonstrates the effect of the modified generation 1.5 dendrimer onIL-1β production. Production was inhibited with treatment of the assayedcompounds. The potency of the dendrimers was demonstrated to increasewith dendrimers prepared using increasing amounts of EDC in the reactionmixtures.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

APPENDIX TO PATENT APPLICATION

TABLE 3 IC₅₀ Constants (μg/ml) for Inhibition of MMP Gen 1.5 Gen 1.5 Gen1.5 Gen 2.5 Gen 2.5 Gen 2.5 1B 1C 1D 2B 2C 2D (serum (serum (serum(serum (serum (serum Free) Free) Free) Free) Free) Free) dd-NAU dd-NAUdd-NAU dd-NAU dd-NAU dd-NAU MMP-1 7.3 6.0 5.0 18.0 6.9 6.0 MMP-3 18.08.0 7.0 29.0 9.0 8.0 MMP-13 3.0 1.7 2.3 8.2 2.4 3.2

TABLE 4 IC₅₀ Constants for Inhibition of MMP Activity NAU Gen 3.5 Gen3.5 Gen 1.5 Gen 1.5 Gen 1.5 Gen 2.5 Gen 2.5 Gen 2.5 Dendrimer A1 B1 1B1C 1D 2B 2C 2D (μg/ml) (serum (serum (serum (serum (serum (serum (serum(serum (Gold Free) Free) Free) Free) Free) Free) Free) Free) Standard)dd-gln-NAU dd-NAU dd-NAU dd-NAU dd-NAU dd-NAU dd-NAU dd-NAU MMP-1 16 1.21.5 2.4 2.0 1.7 2.8 1.0 0.9 MMP-3 17 1.0 1.5 6.0 2.7 1.5 4.5 1.4 1.2MMP-13 10 1.0 1.2 1.0 0.6 0.8 1.3 0.4 0.5

TABLE 5 IC₅₀ Constants (μM) for Inhibition of Pro-inflammatory CytokinesGen 1.5 Gen 1.5 Gen 1.5 Gen 2.5 Gen 2.5 Gen 2.5 1B 1C 1D 2B 2C 2D dd-NAUdd-NAU dd-NAU dd-NAU dd-NAU dd-NAU IL-1β 3.3 2.5 2.0 3.0 0.9 0.8 TNF-α7.0 3.7 2.7 4.6 1.5 1.4 IL-6 1.7 0.7 0.7 0.6 0.3 0.1

1. (canceled)
 2. A method of inhibiting a matrix metalloprotease (MMP)or inhibiting production of a matrix metalloprotease (MMP), the methodcomprising administering to a subject an effective amount of adendrimer, said dendrimer comprising a plurality of branched repeatingunits radiating out from a central atom, multifunctional moiety, orcluster of atoms, wherein each repeating unit comprises a terminalmoiety, and wherein at least about 5% of the terminal moieties include aurea of formula (I)

wherein each R¹ and R² is independently C₁-C₆ alkyl, C₁-C₆ alkenyl,cyclyl, aryl, heterocyclyl, heteroaryl, cyclylalkyl, arylalkyl, orheterocyclylalkyl, heteroarylalkyl; any of which are optionallysubstituted by C₁-C₆ alkyl, amino, C₁-C₆ alkylamino, C₁-C₆ dialkylamino,hydroxy, C₁-C₆ alkoxy, and wherein either of R¹ or R² independentlyoptionally have a net charge;

indicates the point of attachment of the terminal moiety of formula (I)to the dendrimer; and wherein less than about 12% of the terminalmoieties are covalently bound to a saccharide moiety.
 3. The method ofclaim 2, wherein less than about 10% of the terminal moieties arecovalently bound to a saccharide moiety.
 4. The method of claim 2,wherein either of R¹ or R² has a net positive charge.
 5. The method ofclaim 2, wherein R¹ and R² are both cyclohexyl.
 6. The method of claim2, wherein R¹ and R² are both isopropyl.
 7. The method of claim 2,wherein one of R¹ and R² is ethyl and the other of R¹ and R² isdimethylaminopropyl.
 8. The method of claim 2, wherein one of R¹ and R²is cyclohexyl and the other of R¹ and R² is morpholinoethyl.
 9. Themethod of claim 2, wherein each of R¹ and R² is independently C₁-C₆alkyl, and one of R¹ and R² is substituted by C₁-C₆ dialkylamino and hasa positive net charge.
 10. The method of claim 2, wherein a terminalmoiety includes a urea of formula (I′)


11. The method of claim 2, wherein a terminal moiety includes a urea offormula (I″)


12. The method of claim 2, wherein at least about 5% of the terminalmoieties include a urea of formula (I′), formula (I″), or a combinationthereof.
 13. The method of claim 2, wherein the dendrimer is selectedfrom the group consisting of a polyamidoamine dendrimer, a polypropylenedendrimer, a polyethyleneimine dendrimer, a carbohydrate baseddendrimer, a peptide based dendrimer, a glycopeptide dendrimer, a metalcontaining dendrimer, a poly aryl amine dendrimer, a polyamidedendrimer, a poly (alkyl amine) dendrimer, a polyamido alcoholdendrimer, a cyano dendrimer, a polyether dendrimer, a polythioetherdendrimer, a polysiloxane dendrimer, a dendritic aryl ester, aperchlorinated dendrimer, a catalytic center containing dendrimer, asilicon containing dendrimer, a phosphorus containing dendrimer, and ahydrocarbon dendrimer.
 14. The method of claim 13, wherein the dendrimeris a polyamidoamine dendrimer.
 15. The method of claim 2, wherein atleast about 80% of the terminal moieties are terminated with acarboxylate.
 16. The method of claim 15, wherein the dendrimer is apolyamidoamine dendrimer.
 17. The method of claim 2, wherein thedendrimer is a polyamidoamine dendrimer, and wherein at least about 50%of the terminal moieties are terminated with a carboxylate group. 18.The method of claim 2, wherein the dendrimer is a polyamidoaminedendrimer of generation 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, or9.5.
 19. The method of claim 2, wherein the dendrimer is apolyamidoamine dendrimer of generation 0.5, 1.5, 2.5, or 3.5 with acarboxylate terminal moiety.
 20. The method of claim 2, wherein thedendrimer of formula (I) is made by reacting one or more terminalmoieties of a starting dendrimer with a carbodiimide of formula (II)R¹—N═C═N—R², wherein R¹ and R² are defined as in claim 1, to provide thedendrimer of claim 1 formula (I).
 21. The method of claim 2, wherein thematrix metalloprotease (MMP) is selected from the group consisting ofMMP-1, MMP-3 and MMP-13.